Clustering and immobilization of membrane proteins are important in creating and maintaining the polarized epithelial cell phenotype. We have found that newly-synthesized molecules of a glycosylphosphatidylinositol(gpi)-anchored protein, gD-1-DAF, are immobile and clustered when they first reach the apical surface of polarized MDCK cells but are neither immobile nor clustered upon reaching the surface of mutant, non-sorting MDCK. We will investigate the way in which clustering of gpi-anchored proteins functions in the targeting of these proteins to the apical surface of polarized epithelial cells. Clustering and immobilization of gpi-anchored proteins will be probed in plasma membranes, transport vesicles, Golgi-derived vesicles and liposomes and will be compared between normal and sorting-defective cells. Three different fluorescence physical techniques will be used for these probes, FPR, to measure lateral diffusion, RET, to measure molecular proximity and PFD to measure rotational diffusion. Biochemical and immunochemical techniques will complement the fluorescence techniques. The same approach will be used for some transmembrane-anchored apical membrane proteins, allowing comparison of requirements for sorting and targeting of two structurally different classes of membrane proteins.